NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an antivirulence target

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA

Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host

The genetic basis of cadmium resistance of Burkholderia cenocepacia

Burkholderia species are highly resistant to heavy metals (HMs), yet their resistance mechanisms are largely unknown. In this study we screened 5000 mini-Tn5 transposon insertion mutants of Burkholderia cenocepacia H111 for loss of cadmium tolerance. Of the four genes identified three affected outer membrane biogenesis and integrity or DNA repair. The fourth gene, BCAE0587, encoded a P1-type ATPase belonging to the CadA family of HM exporters. CadA-deficient strains lost the ability to grow in the presence of cadmium, zinc and lead, whereas resistance to nickel, copper and cobalt was not affected. Expression studies using a transcriptional fusion of the cadA promoter to gfp confirmed this specificity, as induction was only observed in presence of cadmium, zinc and lead. The promoter activity was found to be highest at neutral pH with an activation threshold of 30 nM cadmium. Inoculation of the HM-hyperaccumulating plant Arabidopsis halleri with a RFP-marked derivative of B. cenocepacia H111 containing the PcadA–gfp fusion demonstrated the applicability of this biosensor for monitoring cadmium at the single cell level in a natural environment

Effects of Bag Mask Ventilation and Advanced Airway Management on Adherence to Ventilation Recommendations and Chest Compression Fraction: A Prospective Randomized Simulator-Based Trial

The role of advanced airway management (AAM) in cardiopulmonary resuscitation (CPR) is currently debated as observational studies reported better outcomes after bag-mask ventilation (BMV), and the only prospective randomized trial was inconclusive. Adherence to CPR guidelines ventilation recommendations is unknown and difficult to assess in clinical trials. This study compared AAM and BMV with regard to adherence to ventilation recommendations and chest compression fractions in simulated cardiac arrests. A total of 154 teams of 3–4 physicians were randomized to perform CPR with resuscitation equipment restricting airway management to BMV only or equipment allowing for all forms of AAM. BMV teams ventilated 6 ± 6/min and AAM teams 19 ± 8/min (range 3–42/min; p < 0.0001 vs. BMV). 68/78 BMV teams and 23/71 AAM teams adhered to the ventilation recommendations (p < 0.0001). BMV teams had lower compression fractions than AAM teams (78 ± 7% vs. 86 ± 6%, p < 0.0001) resulting entirely from higher no-flow times for ventilation (9 ± 4% vs. 3 ± 3 %; p < 0.0001). Compared to BMV, AAM leads to significant hyperventilation and lower adherence to ventilation recommendations but favourable compression fractions. The cumulative effect of deviations from ventilation recommendations has the potential to blur findings in clinical trials

LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants▿

Pseudomonas aeruginosa possesses three quorum-sensing (QS) systems which are key in the expression of a large number of genes, including many virulence factors. Most studies of QS in P. aeruginosa have been performed in clinical isolates and have therefore focused on its role in pathogenicity. P. aeruginosa, however, is regarded as a ubiquitous organism capable of colonizing many different environments and also of establishing beneficial associations with plants. In this study we examined the role of the two N-acyl homoserine lactone systems known as RhlI/R and LasI/R in the environmental rice rhizosphere isolate P. aeruginosa PUPa3. Both the Rhl and Las systems are involved in the regulation of plant growth-promoting traits. The environmental P. aeruginosa PUPa3 is pathogenic in two nonmammalian infection models, and only the double las rhl mutants are attenuated for virulence. In fact it was established that the two QS systems are not hierarchically organized and that they are both important for the colonization of the rice rhizosphere. This is an in-depth genetic and molecular study of QS in an environmental P. aeruginosa strain and highlights several differences with QS regulation in the clinical isolate PAO1

The Impact of Withdrawn vs. Agitated Relatives during Resuscitation on Team Workload: A Single-Center Randomised Simulation-Based Study

Background: Guidelines recommend that relatives be present during cardiopulmonary resuscitation (CPR). This randomised trial investigated the effects of two different behaviour patterns of relatives on rescuers’ perceived stress and quality of CPR. Material and methods: Teams of three to four physicians were randomised to perform CPR in the presence of no relatives (control group), a withdrawn relative, or an agitated relative, played by actors according to a scripted role, and to three different models of leadership (randomly determined by the team or tutor or left open). The scenarios were video-recorded. Hands-on time was primary, and the secondary outcomes comprised compliance to CPR algorithms, perceived workload, and the influence of leadership. Results: 1229 physicians randomised to 366 teams took part. The presence of a relative did not affect hands-on time (91% [87–93] vs. 92% [88–94] for “withdrawn” and 92 [88–93] for “agitated” relatives; p = 0.15). The teams interacted significantly less with a “withdrawn” than with an “agitated” relative (11 [7–16]% vs. 23 [15–30]% of the time spent for resuscitation, p < 0.01). The teams confronted with an “agitated” relative showed more unsafe defibrillations, higher ventilation rates, and a delay in starting CPR (all p < 0.05 vs. control). The presence of a relative increased frustration, effort, and perceived temporal demands (all <0.05 compared to control); in addition, an “agitated” relative increased mental demands and total task load (both p < 0.05 compared to “withdrawn” and control group). The type of leadership condition did not show any effects. Conclusions: Interaction with a relative accounted for up to 25% of resuscitation time. Whereas the presence of a relative per se increased the task load in different domains, only the presence of an “agitated” relative had a marginal detrimental effect on CPR quality (GERMAN study registers number DRKS00024761)